Interleukin-12 (IL-12) is an inflammatory cytokine that is produced in response to infection by a variety of cells of the immune system, including phagocytic cells, B cells and activated dendritic cells (Colombo and Trinchieri (2002), Cytokine & Growth Factor Reviews, 13: 155-168). IL-12 plays an essential role in mediating the interaction of the innate and adaptive arms of the immune system, acting on T-cells and natural killer (NK) cells, enhancing the proliferation and activity of cytotoxic lymphocytes and the production of other inflammatory cytokines, especially interferon-γ (IFN-γ).
IL-12 is a heterodimeric molecule composed of an α-chain (the p35 subunit, IL-12p35) and a β-chain (the p40 subunit, IL-12p40) covalently linked by a disulfide bridge to form the biologically active 74 kDa heterodimer. Amino acid sequences of IL-12p35 and IL-12p40 of a mature (wild-type) human IL-12 are depicted in FIGS. 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2), respectively.
Interleukin-23 (IL-23) is a disulfide-bridged heterodimeric molecule closely related to IL-12, in that it has the same chain IL-12p40 as IL-12, but a unique a chain (the p19 subunit, IL-23p19) (Oppmann et al., (2000), Immunity, 13: 715-725). Like IL-12, IL-23 is produced by phagocytic cells and activated dendritic cells, and is believed to be involved in the recruitment and activation of a range of inflammatory cells (Langrish et al., (2004) Immunol. Rev., 202: 96-105). The amino acid sequence of IL-23p19 of a mature human IL-23 is depicted in FIG. 3 (SEQ ID NO:3).
For immune cells to secrete biologically active IL-12 or IL-23 heterodimers, concomitant expression of the α and β subunits in the same cell is required. Secretion by immune cells of the IL-12p35 or IL-23p19 alone has not been observed, whereas cells that produce the biologically active IL-12 or IL-23 heterodimer secrete the p40 subunit in free form in 10 to 100-fold excess over the heterodimer (D'Andrea et al. (1992), J. Exp. Med., 176: 1387-98, Oppmann et al. (2000), Immunity, 13: 715-725). In addition, it has been observed in the mouse that, even in the absence of an α subunit, cells may produce a biologically active IL-12p40 homodimer (Hikawa et al. (2004), Neuroscience, 129: 75-83).
The presence of endogenous IL-12 has been shown to be necessary for immunological resistance to a broad array of pathogens, as well as to transplanted and chemically induced tumors (Gateley et al. (1998), Annu. Rev. Immunol., 16: 495-521). IL-12 has been demonstrated to have a potent anti-tumor activity based upon the induction of IFN-γ and the activation of effector cells such as CD8+ T-cells and NK cells (Brunda et al. (1993), J. Exp. Med., 178: 1223-30). As a result of its demonstrated anti-tumor activity, IL-12 has been tested in human clinical trials as an immunotherapeutic agent for the treatment of a wide variety of cancers (Atkins et al. (1997), Clin. Cancer Res., 3: 409-17; Gollob et al. (2000), Clin. Cancer Res., 6: 1678-92; and Hurteau et al. (2001), Gynecol. Oncol., 82: 7-10), including renal cancer, colon cancer, ovarian cancer, melanoma and T-cell lymphoma, and as an adjuvant for cancer vaccines (Lee et al. (2001), J. Clin. Oncol. 19: 3836-47).
For IL-12 or IL-23, production of the recombinant protein in its correctly folded and biologically active, heterodimeric form, requires the concurrent expression of both the α subunit and IL-12p40 in the producing cell line. The purified recombinant protein, however, exhibits a degree of heterogeneity resulting from proteolytic cleavage in the C-terminal region of the IL-12p40. The instability of the IL-12 or IL-23 protein can give rise to problems in its production and clinical use as a therapeutic agent. Therefore, there is a need in the art for improved recombinant IL-12 or IL-23 variants that yield a homogeneous protein more resistant to proteolytic cleavage.